Fabrication And Utility Evaluation Of Self-assembled Amphiphilic Delivery Systems Co-loaded With Egg White Derived Peptide And Curcumin

This research was supported by the National Natural Science Foundation of China（32101941）.Over the past few decades,how to make full use of food-derived bioactive substances to regulate physiological functions has a hot issue of concern in food nutrition and human health industry.Meanwhile,more and more research has shown that food-derived hydrophilic and hydrophobic bioactive substances are synergistic in multi-dimensional nutrition improvement and physiological regulation（1+1>2）,which undoubtedly brings an unprecedented opportunity to improve the current multi-factor,multi-symptom,and frequent subhealth status.However,most food-derived hydrophilic and hydrophobic active substances are generally restricted by poor physicochemical stability,processing applicability,system compatibility,and oral bioavailability,which make it difficult to combine bioactive components with different polarities in a single system.Fortunately,the development of the layer-by-layer self-assembly concept has provided the possibility for the steady-state regulation and comprehensive utilization of multiple bioactive components.In addition,the co-delivery research on amphiphilic nutraceuticals（hydrophilic and hydrophobic）is still in its infancy,and the conventional amphiphilic delivery systems are constrained by numerous application bottlenecks,such as low overall loading capacity（≤10%）,ambiguous mechanism of various components,unsatisfactory absorption efficiency of small intestinal,and limited oral bioavailability.Therefore,with the typical hydrophilic EWDP and hydrophobic curcumin as nutraceuticals,the present study aimed to rationally fabricate multi-dimensional amphiphilic delivery systems via layer-by-layer self-assembly.Besides,diverse evaluation methods（e.g.,cytology,zoology,and physical chemistry）comprehensively evaluated the relationship between the colloidal properties and transmembrane co-transport efficiency of nutraceuticals（EWDP-cur）.Moreover,the interaction mechanism between multiple components within the delivery systems was elucidated（especially for the key role of EWDP）.The co-loading properties of different amphiphilic delivery systems on nutraceuticals were revealed at multiple dimensions,including processability,physicochemical stability,bioactivity,and oral bioavailability.Overall,this study provided an important scientific basis and theoretical foundation for the co-delivery and high-value application expansion of food-derived hydrophilic and hydrophobic active substances in the human health field.The main contents and conclusions of this research were as follows.（1）Using alkaline protease-treated egg white protein hydrolysate（<1 k Da）as the raw material,the EWDP sequences were initially screened and identified by HPLC-MS/MS and typical amino acid matching.Afterward,the antioxidant activity of EWDP was comprehensively evaluated based on in vitro chemistry and the 650μM H₂O₂-induced oxidative damage model of He La cells.The key parameters included the free radical scavenging activity（ABTS/ORAC）,cell viability,intracellular antioxidant enzymes（SOD/CAT）activity,and lipid metabolite（MDA）.Results indicated that three EWDP sequences with superior antioxidant activity comparable to the positive control（Trolox）were successfully screened and ranked as CYST>TCNP>YSRQC.This study has laid a solid foundation for the subsequent delivery systems.（2）Usingβ-CD as the amphiphilic core and HTCC as the hydrophilic shell,this study has successfully fabricated a highly-stable amphiphilic delivery system（HTCC-EWDP-β-CD-cur）co-loaded with the above-identified EWDP（CYST）and curcumin via layer-by-layer self-assembly.Results indicated that this novel delivery system showed a compact and homogeneous nano-spherical structure with a relatively steady particle size（153-216 nm）under different p H conditions（2.0-7.0）.Besides,the resulting NPs could effectively co-load EWDP（EC:20.6-28.2%）and curcumin（EC:7.29-7.61%）with excellent colloidal properties and physicochemical stability at p H2.0-7.0.As illustrated by the physicochemical data（FTIR,XPS,1H-NMR,DSC,XRD）,EWDP（both core nutraceuticals and shell material）could cooperate with HTCC to facilitate the curcumin molecules into the deeper hydrophobic cavity ofβ-CD,which further enhanced the overall colloidal properties,and aqueous solubility of the NPs.Additionally,this delivery system could achieve efficient oral co-delivery of nutraceuticals,and exhibit excellent synergistic effects in terms of the controlled release,bioactivities（antioxidant and He La cell growth inhibitory activity）,and absorption properties of nutraceuticals（Caco-2 cell transport efficiency and in vivo bioavailability in BALB/c mice）.This study might break the traditional loading capacity limitation of nutraceuticals（≤10%）.（3）The p H-responsive amphiphilic delivery system co-loaded with EWDP（CYST）and curcumin（CMCS-EWDP-γ-CD-cur）were successfully constructed based on the hydrophilic CMCS shell and amphiphilicγ-CD core.Results implied that this novel oral delivery system showed excellent p H-responsive（2.0-7.0）colloidal properties with a typical nano-spheric structure（172 nm,p H 7.0）.The main driving force for the preferable co-load of EWDP（EC:7.0-12.2%）and curcumin（EC:4.15-4.35%）relied on the complex hydrogen bonding network and hydrophobic interaction.Besides,the NPs showed admirable co-delivery effects with the characteristic controlled release properties and ideal oral bioavailability enhancement.Furthermore,the physicochemical characterization results indicated that EWDP could cooperate with CMCS to modulate the spatial conformation and hydrophobic cavities ofγ-CD molecules in the form of‘affinity reagents’or‘cross-linking agents’with preferable co-loading effects（e.g.,synergistic antioxidant,cellular transport and absorption,gastrointestinal stability,in vivo bioavailability）.These results initially verified the dual functional properties of EWDP（nutrition and processing）,which could provide an enlightening perspective for the co-delivery of food-derived hydrophilic and hydrophobic nutraceuticals.（4）To further exploit the dual functional properties（nutrition and processing）of EWDP,γ-CD-SA was prepared by the tendentious esterification of the hydroxyl group at the C6 position.Then,this study combinedγ-CD-SA and HTCC to facilitate the self-assembly behavior of EWDP within the intestine-targeted amphiphilic delivery system（HTCC-EWDP-γ-CD-SA-cur）through hydrogen bonding and electrostatic interactions.Compared with the unmodified delivery system（HTCC-EWDP-γ-CD-cur）,HTCC-EWDP-γ-CD-SA-cur exhibited the preferable p H-responsive size-controlled properties（particle size:829-380 nm;PDI:0.271-0.175）,and a more uniform and denser nanosphere structure,which could more efficiently co-load EWDP（EC:15.6-24.8%）and curcumin（EC:8.23-8.51%）under multi-p H conditions（2.0-7.0）.Besides,the amphiphilic delivery system exhibited the significant co-loading improvement in terms of processability（storage stability and aqueous solubility）,absorption properties（transmembrane transport properties,bioaccessibility,and antioxidant activity）,and intestinal biodistribution properties.In addition,the transmembrane transport of the NPs mainly relied on the macropinocytosis pathway,which could partially escape the lysosomal degradation to promote the deeper recycling and target delivery of active substances.Notably,the key points in tailoring the actual colloidal properties and oral bioavailability of the delivery system are the co-assembly extent and stabilization level of EWDP,rather than the addition of EWDP or particle size.Related results further reaffirmed the key role of EWDP（both nutrition and processing properties）in the co-encapsulation,co-transportation,and co-delivery of the delivery systems.（5）Based on the egg white protein and corn oil,this study aimed to construct an emulsion-based delivery system co-loaded with curcumin through high-pressure homogenization.With the aid of mass spectrometry,spectroscopy,interface science and digestion kinetics characterization,this study illustrated the effects of glycation（D-ribose）at the lysine residues on the fibrous aggregates,and interface activity（interfacial tension and surface hydrophobicity）of EWGP.Results also indicated that glycation could effectively facilitate the cross-linking extent of interfacial particle network and then regulate the multiple processabilities（e.g.,emulsification,rheology,and storage stability）and nutrient delivery（e.g.,curcumin bioaccessibility,EWDP release properties,lipid digestibility）.Meanwhile,glycation could enhance the overall gastrointestinal digestive tolerance of the delivery system through lysine recognition barriers and spatial repulsion,resulting in the accumulation of peptides with the potential DPP-IV inhibitory activity and larger metabolites（e.g.,long-chain non-ionized FFA or protein fragments with M_W>10 k Da）.Remarkably,these characteristic metabolites might participate in the micellization of hydrophobic nutraceuticals like curcumin,thereby conferring excellent controlled release characteristics to the delivery system.This study could provide a novel insight into the controlled self-release of EWDP and multi-component synergistic homeostasis mechanism,which was expected to fundamentally break the traditional loading capacity limitation of nutraceuticals within delivery systems.